This proposal addresses the problem of bias in the expression profiling of microRNAs (miRNAs) and other small RNAs by next-generation sequencing (NGS). Because dysregulation of miRNA expression has been implicated in cancer and certain other diseases, accurate expression profiling of all miRNA sequences is important for understanding miRNA biology and for development of new biomarkers and therapeutic targets. NGS is currently the most comprehensive approach for digital gene expression profiling, and is used for the discovery of novel miRNA sequences, identification of sequence variants, and the quantification of known miRNAs. Unlike other expression profiling platforms such as microarrays or RT-qPCR, NGS combines unlimited multiplexing capability, single-molecule sensitivity, a superior dynamic range, and true sequence specificity without requiring prior knowledge of miRNA sequences. However, NGS expression profiling data underestimate the amount of many miRNAs in a sample by as much as 10,000-fold. Knowledge of the absolute abundances in samples, and not just the relative changes between samples, is important for reliable identification of miRNAs as biomarkers or drug-target candidates. In NGS, the enzymatic ligation of adapters to the RNA ends is the step where most of the bias in miRNA quantification occurs. The major factors contributing to this ligation bias are intramolecular folding of the miRNAs and intermolecular folding between miRNAs and the adapters, which affect the ability of ligation enzymes to access and ligate the miRNA ends. Thus there is a need for new, more accurate methods, and most previous small RNA profiling experiments should be re- evaluated. To address these problems, we propose a new approach, miR-ACS (miRNA-Adapter Circularization and Sequencing), for preparing miRNA sequencing libraries. Key features of miR-ACS include (i) ligation of miRNAs with only a single adapter; (ii) circularization of the miRNA-adapter ligation product; (iii) elimination of adapter species that are not ligated to miRNAs; and (iv) RT-PCR amplification of the circular miRNA adapter adduct followed by gel-purification of amplicons suitable for NGS sequencing. MiR-ACS has the potential to essentially eliminate miRNA sequencing bias and significantly reduce the number of irrelevant miRNAs sequencing reads (allowing the sequencing of more samples in parallel). These features may also reduce cost and increase the throughput of NGS sequencing. Although miR-ACS is applicable to expression profiling of miRNA or other small RNAs in any sample, for proof-of-concept we focus on samples associated with prostate cancer.